Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms.

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. Graphical abstract ᅟ.

Skin permeation and retention of topical bead formulation containing tranexamic acid.

The objective of this study is to develop a topical bead formulation of tranexamic acid (TA) which can be used concomitantly with laser treatment. The bead formulation of TA (TAB) was successfully prepared by fluidized bed drying method. Physicochemical properties of the TAB were evaluated in terms of chemical stability of TA and differential scanning calorimetry. TA in the bead was stable up to six months at 25°C and existed as amorphous state. In vitro skin permeation and in vivo skin retention of TA in the beads were significantly higher compared to a commercial product. When the bead was dissolved into distilled water and applied concomitantly with laser treatment, the amount of TA retained in the skin in the in vivo study was inversely proportional to the energy levels of laser treatment, indicating absorption into subcutaneous tissue and drainage to systemic circulation. Therefore, when laser treatment is used concomitantly with TAB, energy level should be very carefully monitored to avoid possible adverse events associated with systemic side effects of TA.

In vitro effects of an extracorporeal membrane oxygenation circuit on the sequestration of ϵ-aminocaproic acid.

OBJECTIVE: To assess the in vitro effects of drug sequestration in extracorporeal membrane oxygenation (ECMO) on ϵ-aminocaproic acid (EACA) concentrations. METHODS AND DESIGN: This in vitro study will determine changes in EACA concentration over time in ECMO circuits. A pediatric dose of 2,500 mg was administered to whole expired blood in the simulated pediatric ECMO circuit. Blood samples were collected at 0, 30, 60, 360 and 1440-minute intervals after initial administration equilibration from three different sites of the circuit: pre-oxygenator (PRE), post-oxygenator (POST) and PVC tubing (PVC) to determine the predominant site of drug loss. The circuit was maintained for two consecutive days with a re-dose at 24 hours to establish a comparison between unsaturated (New) and saturated (Old) oxygenator membranes. Comparisons between sample sites, sample times and New versus Old membranes were statistically analyzed by a linear mixed-effects model with significance defined as a p-value <0.05. RESULTS: There were no significant differences in EACA concentration with respect to sample site, with PRE and POST samples demonstrating respective mean differences of 0.30 mg/ml and 0.34 mg/ml as compared to PVC, resulting in non-significant p-values of 0.373 [95% CI (-0.37, 0.98)] and 0.324 [95% CI (-0.34, 1.01)], respectively. The comparison of New vs. Old ECMO circuits resulted in non-significant changes from baseline, with a mean difference of 0.50 mg/ml, 95% CI (-0.65, 1.65), p=0.315. CONCLUSION: The findings of this study did not show any significant changes in drug concentration that can be attributed to sequestration within the ECMO circuit. Mean concentrations between ECMO circuit sample sites did not differ significantly. Comparison between New and Old circuits also did not differ significantly in the change from baseline concentration over time. Sequestration within ECMO circuits appears not to be a considerable factor for EACA administration.

[The unresolved issue of false-positive D-dimer results in the diagnostic workup of pulmonary embolism]. / Diagnostic d'embolie pulmonaire: problématique résiduelle des D-dimères faussement positifs.

The unresolved issue of false-positive D-dimer results in the diagnostic workup of pulmonary embolism Pulmonary embolism (PE) remains a difficult diagnosis as it lacks specific symptoms and clinical signs. After the determination of the pretest PE probability by a validated clinical score, D-dimers (DD) is the initial blood test in the majority of patients whose probability is low or intermediate. The low specificity of DD results in a high number of false-positives that then require thoracic angio-CT. A new clinical decision rule, called the Pulmonary Embolism Rule-out criteria (PERC), identifies patients at such low risk that PE can be safely ruled-out without a DD test. Its safety has been confirmed in US emergency departments, but retrospective European studies showed that it would lead to 5-7% of undiagnosed PE. Alternative strategies are needed to reduce the proportion of false-positive DD results.

[Emergency department point of care testing: what are the benefits and for which patients?]. / Laboratoire délocalisé aux urgences: quels bénéfices pour quels patients?

Laboratory tests contribute to patient length of stay in the emergency department. Therefore, rapid tests performed at the bedside (POCT or point of care testing) are attractive because they allow the emergency physician to obtain immediate biological, diagnostic and/or prognostic data. Userfriendly and with validated analytical performance, POCT have the potential to reduce laboratory time, patient length of stay and time to treatment or disposition. The expected benefit from POCT implementation will depend on the type of patients involved (inpatient or outpatient), their clinical condition and their overall care. Furthermore, logistical and economic implications should also be taken into account.

Spectrophotometric determination of tranexamic acid using vanillin.

A new spectrophotometric method has been examined for the determination of the tranexamic acid (TA) by derivatization with vanillin (VAN). The molar absorptivity of TA was calculated 25,160 L x mol(-1) x cm(-1) at lambdamax 354 nm and obeyed the Beer's law within 0.5-2.5 microg x mL(-1). The color reaction was highly stable and did not show any change in absorbance up to 24 h. The method was applied for the analysis of TA from capsules, injections and tooth pastes. The amounts of TA found in capsules, injections and tooth pastes of various pharmaceutical companies were observed with 249.0-250.9 mg/capsule, 249.3-250.7 mg/injection and 0.048%-0.049% in tooth pastes with relative standard deviation (RSD) 0.2%-5.0% (n = 3).

[Clinical and radiological findings in patients diagnosed pulmonary thromboembolism by pulmonary computerized tomography angiography]. / Pulmoner bilgisayarli tomografi anjiyografi ile pulmoner tromboemboli tanisi konulan hastalarda klinik ve radyolojik bulgular.

Pulmonary computed tomography angiography (PCTA) is the initial imaging test for the diagnosis of pulmonary thromboembolism (PTE). In the study, it was aimed to determine the clinical, radiological findings in patients diagnosed PTE by PCTA, to investigate the relationship between the thrombus localisation and the clinical, laboratory parameters. 172 patients diagnosed PTE by PCTA between 2004 and 2007 were included in the study. The clinical, laboratory parameters, thrombus localisation were evaluated. Mean age (female/male: 99/73) was 58.27 +/- 15.11, mean Wells score was 2.99 +/- 2.40. 39.5% (n= 68) of patients had low risk, 50.6% (n= 87) intermediate, 9.9% (n= 17) high risk. The most common comorbidities were cardiovascular diseases (n= 46, 26.7%), COPD (n= 26,15%). Recent operation history (n= 47, 27.3%), immobilisation (n= 37, 21.5%) were the most frequent risk factors. Dyspnea (89%), chest pain (59.9%) were the most common complaints. Deep venous thrombosis was detected by Doppler USG in 56.4% of patients. The most common site of thrombus was the right lower lobe artery (44.2%). In 30% of patients, the most proximal level of thrombus was the main pulmonary arteries (MPA). Mean age of patients with MPA thrombus (61.96 +/- 14.47), was higher than patients with distal thrombus (56.62 +/- 15.16, p= 0.03). Patients with the recent operation history (41% vs. 21%, p= 0.009), cancer (24% vs. 2.5%, p< 0.001) had higher rates of MPA thrombus. In patients presented with syncope,16.9% of them had a MPA thrombus compared to others having 3.3% rate of other thrombus localisations (p= 0.004). Mean Wells score in patients with MPA thrombus was higher compared to others (3.59 +/- 2.38/2.72 +/- 2.37, p= 0.02), however it didn't differ the extent of proximal thrombus between low, intermediate and high risk patients. The mean level of D-dimer was not different between patients with MPA thrombus and the others. D-dimer level was significantly higher in patients with thrombus localized at truncus pulmonalis (1357 microg/mL vs. 724 microg/mL). There was no significant difference between Doppler USG positive and negative patients for DVT. In conclusion, it was determined that the thrombus was at MPA in one third of the patients, a significant relationship between the presence of the recent operation, cancer history and syncope with MPA thrombus. In patients with a thrombus at truncus pulmonalis, D-dimer levels were higher.

Melatonin administration increased plasminogen activator activity in ram spermatozoa.

The aim of the present study was to investigate the effect of melatonin on plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in ram spermatozoa and seminal plasma, in correlation with changes in blood testosterone. Melatonin implants (18 mg) were placed subcutaneously in sixteen Chios rams in autumn and spring. Semen samples for spectrophotometrical assays were collected 36 h before the implantation of melatonin and thereafter once a week, for 17 weeks. Blood samples for testosterone assay (RIA) were collected 8h before implantation (one sample/30 min x 7.5 h) and thereafter every 15 days for 105 days after implantation. For each ram, six parameters of testosterone were estimated: mean value, basal level, number of peaks, peak amplitude, peak duration and mean testosterone concentration during peaks. Melatonin implantation during autumn induced an increase in PAA and t-PAI in spermatozoa; melatonin implantation in spring induced an additional increase in u-PAI and PI; no change in PAA, PAI or PI was found in seminal plasma, during autumn or spring. The melatonin-induced increase of PAA, PAI and PI in spermatozoa was in positive correlation with the increase of testosterone mean value, basal level and number of peaks; the increase of testosterone parameters was greater in autumn compared to spring. Changes of PAA, PAI and PI of spermatozoa, under the influence of melatonin, might indicate changes in the fertilizing ability of spermatozoa, since plasminogen activators and their inhibitors are present on the plasma and the outer acrosomal membrane of spermatozoa and are released during the acrosome reaction.

Reduction of contact activation related fibrinolytic activity in factor XII deficient patients. Further evidence for the role of the contact system in fibrinolysis in vivo.

In this study the contribution of activation of the contact system to activation of the fibrinolytic system in vivo was investigated in healthy volunteers and in factor XII deficient patients. The plasminogen activating activity in plasma from healthy volunteers after infusion of desamino D-arginine vasopressin (DDAVP) was only partially blocked (for 77%) with specific antibodies to tissue-type plasminogen activator and urokinase type plasminogen activator. The residual activity could be quenched by a monoclonal antibody that inhibits factor XII activity and was not present in patients with a factor XII deficiency. The formation of plasmin upon the DDAVP stimulus as reflected by circulating plasmin-alpha 2-antiplasmin complexes was lower in factor XII deficient patients than in healthy volunteers. Activation of the contact system occurred after DDAVP infusion in healthy volunteers and was absent in factor XII deficient patients. These results indicate that DDAVP induces a plasminogen activating activity that is partially dependent on activation of the contact system and that contributes to the overall fibrinolytic activity as indicated by the formation of plasmin-alpha 2-antiplasmin complexes. This fibrinolytic activity is impaired in factor XII deficient patients which may explain the occurrence of thromboembolic complications in these patients.

[Diagnosis of pulmonary embolism]. / Diagnostic de l'embolie pulmonaire.

The diagnosis of pulmonary embolism (PE) is challenging because the symptoms are unspecific and an objective confirmation of the disease is required. Clinical symptoms and physical examination can be used to estimate the clinical probability of the disease used to interpret the results of the diagnostic tests. In patients with low or moderate clinical probability most quantitative D-dimer tests allow to exclude PE safely. A positive proximal venous ultrasound enables to confirm PE but the sensitivity is too low to exclude the diagnosis. In patients with a positive D-dimer test, multidetector spiral CT allows to confirm or exclude PE with confidence in most of the cases. Lung scanning is still useful in patients with renal impairment or allergy to contrast medium.

Normal D-dimer levels in emergency department patients suspected of acute pulmonary embolism.

OBJECTIVES: We sought to determine:1) whether normal D-dimer enzyme-linked immunosorbent assay (ELISA) assays predicted the absence of pulmonary embolism (PE) in the high-volume emergency department (ED) of the Brigham and Women's Hospital, and 2) whether ED physicians accepted normal D-dimer levels as confirmation of no PE without further diagnostic testing such as lung scanning, chest computed tomography (CT) scanning, or pulmonary angiography. BACKGROUND: Although the plasma D-dimer ELISA is a sensitive screening test for excluding acute PE, this laboratory marker has not been widely integrated into clinical algorithms such as creatine kinase-MB fraction or troponin testing for acute myocardial infarction. METHODS: We mandated that ED physicians order D-dimer ELISA tests on all patients suspected of acute PE. We reviewed the clinical record of each ED patient initially evaluated for suspected PE during the year 2000. We determined whether additional imaging tests for PE were obtained and whether the final diagnosis was PE. RESULTS: Of 1,106 D-dimer assays, 559 were elevated and 547 were normal. Only 2 of 547 had PE despite a normal D-dimer. The sensitivity of the D-dimer ELISA for acute PE was 96.4% (95% confidence interval [CI]: 87.5% to 99.6%), and the negative predictive value was 99.6% (95% CI: 98.7% to >99.9%). Nevertheless, 24% of patients with normal D-dimers had additional imaging tests for PE. CONCLUSIONS: The D-dimer ELISA has a high negative predictive value for excluding PE. By paying more attention to normal D-dimer results, fewer chest CT scans and lung scans will be required, and improvements may be realized in diagnostic efficiency and cost reduction.

The emergency department utility of Simplify D-dimer to exclude pulmonary embolism in patients with pleuritic chest pain.

STUDY OBJECTIVE: Pleuritic chest pain is a common presenting complaint in the emergency department (ED) and a symptom of pulmonary embolism. Patients with pleuritic chest pain would benefit from a simple and rapid way of screening for pulmonary embolism. The aim of this study is to assess the utility of Simplify D-dimer as a rule-out tool for pulmonary embolism in ED patients with pleuritic chest pain. METHODS: This was a prospective diagnostic study in a large city-center ED. Four hundred twenty-five patients with pleuritic chest pain were prospectively recruited between February 2002 and June 2003. Simplify D-dimer testing was performed on each patient in the ED. All patients followed an independent reference standard diagnostic algorithm for pulmonary embolism. Each patient was followed up clinically for 3 months. RESULTS: The calculated sensitivity of Simplify D-dimer for pulmonary embolism was 81.8% (95% confidence interval [CI] 61.4% to 92.7%), and specificity was 74.2% (95% CI 69.6% to 78.4%). The negative predictive value was 98.6% (95% CI 96.6% to 99.6%), positive predictive value 15.0% (95% CI 9.1% to 22.7%), negative likelihood ratio 0.25 (95% CI 0.10 to 0.52) and positive likelihood ratio 3.17 (95% CI 2.30 to 3.97). The study cohort pretest probability was 5.3%. A negative Simplify result reduced the posttest probability to 1.3% (95% CI 0.5% to 3.4%). CONCLUSION: The Simplify D-dimer is not sufficiently sensitive to exclude the diagnosis of pulmonary embolism in all patients presenting to the ED with pleuritic chest pain.

Fibrinolytic inhibitors in off-pump coronary surgery: a prospective, randomized, double-blind TAP study (tranexamic acid, aprotinin, placebo).

OBJECTIVE: To evaluate and compare hemostatic effects of tranexamic acid vs. aprotinin vs. placebo in off-pump coronary artery bypass (OPCAB) surgery and, in addition, to assess the safety of fibrinolytic inhibitors therapies. METHODS: In a prospective, randomized, double-blind study finally 91 patients undergoing OPCAB were investigated (group A, n=32, tranexamic acid 1g before skin incision and continuously 200mg/h; group B, n=29, aprotinin 1,000,000IU before skin incision and 250,000IU/h; group C, n=30, placebo). RESULTS: Highly significant inter-group differences were found in cumulative blood loss within 4h (geometric means [95% confidence intervals]-group A: 89.3 [72.7, 109.8] mL, group B: 72.3 [49.2, 106.3] mL and group C: 192.3 [151.8, 243.5] mL) (P<0.001), within 8h (group A: 152.1 [120.7, 191.6] mL, group B: 130.3 [88.1, 192.8] mL and group C: 283.8 [226.0, 356.3] mL) (P=0.001), and within 24h postoperatively (group A: 410.3 [337.6, 498.6] mL, group B: 345.8 [256.0, 398.2] mL and group C: 619.8 [524.3, 732.8] mL) (P<0.001). At all time points, placebo group C was significantly distinct from the groups treated with fibrinolytic inhibitors (groups A and B). However, no differences between groups A and B were found. Both mean hemoglobin and hematocrit values 24h postoperatively were different between the groups (P=0.018 and P=0.077, respectively), acheiving the lowest value in group C. Number of re-transfuzed patients was highest in group C, but without statistical significance (either packed red blood cells, P=0.119 or fresh-frozen plasma, P=0.118). We observed one postoperative myocardial infarction in aprotinin treated group B and one temporary postoperative myocardial ischemia in placebo group C, no cerebrovascular or pulmonary embolism was noticed. Treated groups A and B did not demonstrate postoperative increase in mean levels of myocardial enzymes, compared with group C. Significantly higher mean values of D-dimer were found in group C 24h postoperatively (P<0.001). CONCLUSIONS: Both tranexamic acid and aprotinin seem to be similarly effective in the reduction of postoperative blood loss in OPCAB. Tranexamic acid appears to be cost-effective and safe alternative to aprotinin.

Diagnosis of pulmonary embolism by a decision analysis-based strategy including clinical probability, D-dimer levels, and ultrasonography: a management study.

BACKGROUND: Assessment of the clinical probability of pulmonary emboli sm, plasma D-dimer measurement, and lower-limb venous compression ultrasonography have all been advocated in the workup of suspected pulmonary embolism, to minimize the requirement for pulmonary angiography in patients with nondiagnostic lung scans. However, their contribution has not been assessed prospectively. METHODS: Three hundred eight consecutive patients who came to the emergency department with suspected pulmonary embolism were managed according to a diagnostic protocol that included clinical probability assessment, lung scan, and sequential noninvasive tests: plasma D-dimer measurement by enzyme-linked immunosorbent assay (a concentration <500 microgram/L ruled out pulmonary embolism) and lower-limb B-mode venous compression ultrasonography (a positive finding was diagnostic of venous thromboembolism). Patients without pulmonary embolism according to the diagnostic workup did not receive anticoagulant treatment. The safety of this approach was assessed by a 6-month follow-up. RESULTS: of the 308 patients, 106 (34%) had a diagnostic lung scan (normal in 43 and high probability in 63). For the remaining 202 patients, noninvasive workup was diagnostic in 125 (62%). Pulmonary embolism was ruled out by a low clinical probability and a nondiagnostic scan in 48 patients and a D-dimer level less than 500 microgram/L in 53; pulmonary embolism was established by a high clinical probability and a nondiagnostic scan in seven patients and by a finding of a deep vein thrombosis on ultrasonography in 17. Therefore, only 77 of these 202 patients underwent pulmonary angiography (negative in 55; positive in 22). At 6-month follow-up (completed for 99.4% of the study population), only two of the 199 patients in whom the diagnostic protocol had ruled out pulmonary embolism (1.0% [95% confidence interval, 0.1 to 3.6]) had a thromboembolic event (pulmonary embolism, one; deep vein thrombosis, one). CONCLUSIONS: This decision analysis strategy yielded a definitive noninvasive diagnosis in 62% of patients with a nondiagnostic scan and appears to be safe.

Analysis of Prethrombotic States in Patients with Malignant Tumors.

BACKGROUND: This study aimed to investigate the incidence and risk factors for a prethrombotic state in patients with malignant tumors. MATERIALS AND METHODS: Plasma d-dimer (D-D) in patients with malignant tumors was measured. Abnormal rates of D-D and possible risk factors like gender, age, type of tumor, and staging of tumor were analyzed. RESULTS: Of 1,453 patients, 629 demonstrated plasma D-D abnormality (43.3%). The D-D abnormal rate of male patients (n=851, 43.5%) was not statistically significantly different from that for female patients (n=602, 43.0%) (p>0.05). D-D abnormal rate increased with age and was statistically significant among different age groups (p<0.05). Regarding staging of tumor, D-D abnormal rate in patients with phase I was 2.0%, 6.2% in phase II, 47.6% in phase III and 83.1% in phase IV, with statistically significant differences between phase III and II, as well as phase III and IV (p<0.01). CONCLUSIONS: A prethrombotic state was closely related to malignancy of tumors. The risk factors for a prethrombotic state include age and tumor stage.

Fibrin deposition and clearance in chronic granulomatous inflammation: correlation with T-cell function and proteinase inhibitor activity in tissue.

Patterns of fibrin deposition were investigated by immunofluorescence microscopy in livers of thymus intact (TI) and athymic (AT) mice infected with Schistosoma mansoni. Thrombin and fibrinolysis inhibitor activity in tissue extracts also were measured. In TI mice fibrin was detected perivascularly by 6 weeks after infection and at 8 weeks it was found over the granulomas as they developed. Fibrin was cleared from the center of granulomas by 10 weeks. Thrombin inhibitor activity increased at 4 to 6 weeks but declined below control levels later as granulomas formed. Fibronolysis inhibitor activity, on the other hand, peaked at 9 to 12 weeks after infection. In AT mice extensive fibrin deposition was detected in the liver throughout the period when smaller and incomplete granulomas developed. Central clearing did not occur. Thrombin inhibitor activity greatly increased by 8 weeks after infection but fibrinolysis inhibitor activity remained unchanged. These findings suggest that fibrin deposition and firbinolysis are orderly events regulated in the lesions by proteinases and their inhibitors and this seems to be a general tissue reaction in the early stage of chronic granuloma formation. Since local clearance of fibrin in vivo and fibrinolysis inhibitor activity from tissue extracts studied in vitro are more evident in TI mice than in AT mice, it appears that T cell fu;ction is important in modulating the tissue response during granulomatous inflammation.

Lipid-related hemostatic abnormalities in the elderly: imbalance between coagulation and fibrinolysis.

To clarify age-related and lipid-related hemostatic abnormalities in the elderly, we measured the plasma levels of active PAI-1 antigen (aPAI-1), tPA-PAI-1 complex (TPC), plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI), plasmin-alpha 2-PI complex (PIC), and D-dimer, together with the plasma levels of fibrinogen, factor VII (F VII), and thrombin-antithrombin III complex (TAT) and the serum lipid levels in 68 hyperlipidemic and 82 normolipidemic elderly subjects. The aPAI-1 ratio was calculated as aPAI-1/(aPAI-1 + TPC). In the normolipidemic elderly subjects, plasma PIC and D-dimer levels were much higher when compared with healthy young controls, and there was also a decrease in plasma plasminogen and alpha 2-PI levels, an increase in plasma TPC levels, and high plasma F VII and fibrinogen levels. In elderly subjects with type IIb hyperlipidemia, both the plasma aPAI-1 level and the aPAI-1 ratio were significantly increased, while the plasma PIC and D-dimer levels were reduced despite higher plasma F VII, fibrinogen and TAT levels. Both serum total cholesterol and triglyceride levels were correlated positively with plasma F VII and TAT levels and with the TAT/PIC ratio, while only serum triglyceride levels showed a positive correlation with plasma TPC and aPAI-1 levels and with the aPAI-1 ratio. Thus, an increase of fibrinolytic activity appears to occur as part of normal aging to balance the increase of procoagulant activity. However, an imbalance between thrombin activity (increased procoagulant activity) and plasmin activity (hypofibrinolysis) appears to occur in elderly individuals with hyperlipidemia, perhaps resulting in a predisposition to thromboembolic disease.

Homogeneous immunoassay for alpha 2 plasmin inhibitor (alpha 2PI) and alpha 2PI-plasmin complex. Application of a sandwich liposome immune lysis assay (LILA) technique.

The measurement of the alpha 2 plasmin inhibitor (alpha 2PI) and alpha 2PI-plasmin complex is important for a complete understanding of fibrinolytic conditions. Using monoclonal antibodies against alpha 2PI, a sandwich liposome immune lysis assay (LILA) has been used for the quantitation of alpha 2PI and the alpha 2PI-plasmin complex. In the assay system for the measurement of alpha 2PI, anti-alpha 2PI monoclonal antibodies were covalently coupled to liposomes and specific lysis of liposomes was observed when the liposomes were incubated with the alpha 2PI antigen, TNP haptenized second monoclonal antibody against alpha 2PI and complement activating rabbit anti-TNP antibody. The same liposomes and rabbit anti-plasminogen antibody could be used for the homogeneous determination of the alpha 2PI-plasmin complex. The former assay suggests that monoclonal antibodies lacking complement-activating ability can be used in the sandwich LILA technique. The second application suggests that the LILA technique is capable of measuring heterocomplexes. These assays, which involve the same analytical system, are simple, fast and highly sensitive. They are potentially useful in determining the fibrinolytic status of patients.

Changes in human amniotic fluid fibrinolytic inhibitor levels during pregnancy.

The amniotic fluid (AF) when incubated with the patient's own plasma diminishes the lytic activity of the plasma. It is suggested that this inhibition is due to the presence of fibrinolytic inhibitors in the AF. The inhibitors rate increases as pregnancy advances. Evaluating these inhibitors in a group of 65 women before and after the 38th week of pregnancy, a higher rate of fibrinolytic inhibitors is found after the 38th week. The said differences are statistically significant. For the moment it does not seem that the increasing of the inhibitors in the last part of pregnancy might be used as a fetal maturity test.

Partial characterization of a fibrinolytic inhibitor produced by cultured endothelial cells derived from human umbilical vein.

A preliminary characterization of a fibrinolytic inhibitor released by human umbilical vein endothelial cells in primary culture is reported. This molecule of Mr comprised between 2 X 10(5) and 10(6) and of alpha 2 mobility precipitates at 43% ammonium sulphate saturation and is totally adsorbed on Concanavalin A Sepharose 4 B. A possible relationship with alpha macroglobulins is discussed.